The myelopoietic and biochemical relationships of human eosinophils and basophils and their functional interactions in disease are unclear. A fundamental lineage association between these granulocytes is supported by in vitro observations that these cells may differentiate from a common progenitor present in human peripheral blood and bone marrow, and the finding that certain presumably eosinophil-specific membrane and cationic granule proteins are also prominent constituents of basophils, but not other myeloid cells. One of these proteins, the Charcot- Leyden crystal (CLC) protein, a membrane-associated hydrophobic polypeptide with lysophospholipase (lysolecithin acylhydrolase, E.C. activity, is the sole protein component of CLC, and constitutes 7-10% of total cellular protein in these granulocytes. to further elucidate interrelated aspects of the differentiation and functions of eosinophils and basophils, we will investigate the molecular biology of these cells by cloning, sequencing, and studying the regulation of genes coding for a number of their membrane and granule proteins. Focusing initially on the gene for CLC protein, we propose to: 1) clone and sequence the cDNA for CLC protein and isolate and study the organization of a genomic clone, 2) study the regulation of CLC protein gene expression at the mRNA level during myeloid differentiation, 3) study the structure and functions of recombinant CLC protein by investigating the effects of site- specific mutations on post-translational modifications, enzymatic and non-enzymatic activities, subcellular localization, and formation of CLC, and 4) clone and study the genes coding for the eosinophil granule cationic proteins, major basic protein, eosinophil-derived neurotoxin, eosinophil cationic protein, and eosinophil peroxidase in a similar fashion. cDNA corresponding to the CLC and granule cationic proteins will be cloned from expression libraries prepared using mRNA from cells shown to be actively synthesizing the proteins, including cells of the promyelocytic leukemia line HL-60, normal human bone marrow cells, and bone marrow cells from a patient with the hypereosinophilic syndrome. Gene libraries will be screened using monospecific antisera to the proteins and oligonucleotide probes designed from the amino acid sequence of tryptic peptides. These studies aim to enhance our understanding of the biologic activities of CLC and the granule cationic proteins, the hematopoietic relationships of eosinophils and basophils, and their functions in allergic, inflammatory, and related immunologic responses.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Hematology Subcommittee 2 (HEM)
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Beth Israel Deaconess Medical Center
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